CN112409590A - Organic nano assembly for biological imaging of second window in near infrared region and preparation method and application thereof - Google Patents
Organic nano assembly for biological imaging of second window in near infrared region and preparation method and application thereof Download PDFInfo
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- CN112409590A CN112409590A CN202011133706.7A CN202011133706A CN112409590A CN 112409590 A CN112409590 A CN 112409590A CN 202011133706 A CN202011133706 A CN 202011133706A CN 112409590 A CN112409590 A CN 112409590A
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Abstract
The invention belongs to the technical field of biological materials, and particularly relates to an organic nano assembly for biological imaging of a second window in a near infrared region, and a preparation method and application thereof. The organic nano-assembly is a nano-material synthesized based on piperazine thiochromene pentamethine cyanine dye structure, is marked as NIRlyso-NP, and the monomer of the organic nano-assembly mainly comprises three parts: a series of piperazine benzothiopyran pentamethine cyanine dye structures with luminescent properties, flexible chains connected in the middle and terminal functional groups; the material has the characteristics of fluorescent signal activation and target identification, can be used for high signal-to-noise ratio living body biological imaging of a near infrared second window, and provides help for disease detection, diagnosis and fluorescence-guided surgery treatment.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a nano assembly and a preparation method thereof, and application of the nano assembly in near-infrared second window living organism high signal-to-noise ratio imaging and operation navigation.
Background
In recent years, medical imaging technology is widely applied in the medical industry and is an important tool for early diagnosis, monitoring, curative effect evaluation and surgical navigation of diseases. The most clinical imaging techniques at present belong to tomography, such as Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), and ultrasound. These techniques have some limitations: such as CT and PET, can introduce harmful ionizing radiation, limited spatial and temporal resolution of MRI imaging, and the need for functional contrast agents. In contrast, fluorescence imaging technology has great application prospects due to its advantages of real-time dynamics, high resolution, no harmful radiation and the like. For the traditional fluorescent dye in the visible light area, researchers are dedicated to various designs and improvements on the molecular structure, so that the fluorescent dye can generate specific response to a biological marker, and the change of the fluorescence intensity before and after the response is realized; or modifying the molecule with a group with a target recognition function, so that the molecule can have larger enrichment amount in a target region, thereby improving the signal-to-noise ratio of imaging.
For the fluorescent probe with the near-infrared second window (900-. However, currently developed near-infrared two-zone fluorescent probes are limited in types, generally only have a light-emitting characteristic, do not have targeting and activating characteristics, and cannot realize specific lighting on a specific part of an organism, so that the signal-to-noise ratio of imaging is still limited; and the retention time of the probe at the focus part is short, so that the observable time window is limited, and the effect of the probe on in-vivo biological imaging is greatly limited.
Disclosure of Invention
The invention aims to provide a near-infrared second window excited and emitted organic nano assembly with good biocompatibility, high signal-to-noise ratio and good specificity, and a preparation method and application thereof.
The invention provides an organic nano assembly, a nano material synthesized based on piperazine benzothiopyran pentamethine cyanine dye structure is marked as NIRlyso-NP, and a monomer (NIRlyso) mainly comprises three parts: a series of piperazine benzothiopyran pentamethine cyanine dye structures with luminescent properties, flexible chains connected in the middle and terminal functional groups; the monomer has a general structural formula shown in formula (I):
wherein:
x is selected from O or S, R1～R5Can be independently selected from O (CH)2)n1CH3Or (CH)2)n1CH3Alkyl chains, such as H, F, Cl, Br, I atoms; r6Is selected from OCH3Succinimide, maleimide, N3RGD or folic acid, Y is selected from ClO4、PF6、BF4、Cl、Br、I、CF3COO、CF3SO3Or CH3SO3，R7Can be selected from one of the groups shown in the formula (II), wherein n is an integer of 20-50;
the core fluorophore structure of the organic nano assembly is a novel functionalized piperazine benzothiopyran pentamethine cyanine dye structure which is expanded and innovatively designed on the basis of the previous research, the research and the research of a synthetic route are completed, and a substituted piperazine group N (CH) is modified2)2(CH2)2NCH2CHCH, the structure of which is not reported in the literature.
The nanometer assembly synthesized based on the piperazine thiochromene pentamethine cyanine dye structure has the characteristics of fluorescent signal activation and targeted identification, so that the nanometer assembly can be used for high signal-to-noise ratio living body biological imaging of a near-infrared second window, and provides help for disease detection, diagnosis and fluorescence-guided surgery treatment. For example, the fluorescent probe is used for the living body imaging of the mouse with high signal to noise ratio as a near infrared two-region fluorescent probe.
The fluorescence signal activatable characteristic of the organic nano-assembly of the invention is shown as follows:
(1) in a fluorescence quenching state in a water system, the laser does not emit light when irradiated by a 800-1000 nm laser;
(2) when the surfactant is added into the nano assembly system, the structure of the nano assembly is damaged and converted into a fluorescence luminescent state, and the 800-1000 nm laser emits light;
(3) when the nano assembly is phagocytized by cells, the structure of the nano assembly is destroyed and converted into a fluorescence state, and the 800-1000 nm laser emits light under irradiation.
The specific targeting characteristics of the organic nano-assembly are shown as follows:
the end R6 of the assembly can be directly modified with a group with biological target recognition function through chemical bonding, so that the assembly is enriched in a target area through the specific binding function of a target-receptor.
The preparation method of the organic nanometer assembly provided by the invention comprises the following specific steps:
(1) substituted thiophenol, substituted ethyl benzoate and propine substituted piperazine are taken as raw materials, and are subjected to polyphosphoric acid condensation cyclization reaction and Buchwald-Hartwig amination reaction sequentially to obtain substituted benzothiopyran derivatives; the benzothiopyran derivative is further alkylated by a methylation format reagent and is treated by acid to prepare a terminal salt, and the terminal salt and a condensing agent of amylene dialdehyde diphenylamine hydrochloride undergo a Knoevenagel condensation reaction to obtain a series of piperazine benzothiopyran pentamethine cyanine dye structures with a propine structure at the tail end and with luminescent properties;
(2) the dye is further connected with a polyethylene glycol chain with one end modified with an azide group through a click chemical reaction to obtain an amphiphilic monomer structure;
(3) the tail end of the monomer structure is further subjected to bio-orthogonal reaction to obtain a nano assembly;
(4) or further performing an amide reaction to modify the functional biomolecule.
More specifically, the preparation method of the organic nano assembly provided by the invention comprises the following specific steps:
(1) synthesis of monomer (NIRlyso) comprising:
the specific synthetic route is as follows:
the specific synthesis steps are as follows:
(1-1) Synthesis of intermediate 1
Dissolving para-bromo substituted thiophenol (marked as a compound 1) and substituted ethyl phenylacetate (marked as a compound 2) in polyphosphoric acid, and reacting for 1-3 hours at 90-100 ℃; cooling, adding crushed ice to quench the reaction, extracting with dichloromethane, concentrating an organic phase, and separating by column chromatography to obtain an intermediate 5-1; wherein the feeding molar ratio of the compound 1 to the compound 2 to the polyphosphoric acid is 1 (1-1.3) to 10-15;
(1-2) Synthesis of intermediate 2
Under the protection of nitrogen, mixing the intermediate 1, tert-butyloxycarbonylpiperazine (marked as a compound 3), a Buchwald catalyst and inorganic base in a dry solvent, and reacting for 3-12 hours at 80-110 ℃; cooling to room temperature, filtering, concentrating the organic phase, and separating by column chromatography to obtain an intermediate 2; wherein the Buchwald catalyst is a composition which is selected from one of palladium acetate and tris (dibenzylideneacetone) dipalladium and one of 2-dicyclohexyl phosphorus-2 ',4',6 '-triisopropyl biphenyl, 4, 5-bis (diphenylphosphino) -9, 9-dimethyl xanthene and 2-dicyclohexyl phosphine-2' - (N, N-dimethylamine) -biphenyl, and the feeding mole percentage of the Buchwald catalyst is 1-10% of that of the intermediate 5-1; the feeding molar ratio of the intermediate 1, the compound 3 and the inorganic base is 1 (2-5) to 1.2-3, and the inorganic base is selected from one of sodium tert-butoxide, cesium carbonate, potassium carbonate and potassium phosphate; the solvent is selected from one of toluene, dioxane and tetrahydrofuran;
(1-3) Synthesis of intermediate 3
Dissolving the intermediate 2 in a mixed solvent of dichloromethane and trifluoroacetic acid, stirring for 1-5 hours in an ice bath, and after the reaction is finished, spin-drying the organic solvent to obtain a solid, namely an intermediate 3;
(1-4) Synthesis of intermediate 4
Dissolving the intermediate 3, bromopropyne (marked as a compound 4) and potassium carbonate in acetonitrile, reacting for 10-24 hours at 80 ℃, cooling to room temperature, filtering, concentrating an organic phase, and separating by using column chromatography to obtain an intermediate 4; wherein the feeding molar ratio of the intermediate 3 to the compound 4 to the potassium carbonate is 1: (2-5): (2-5);
(1-5) Synthesis of intermediate 5
Dissolving the intermediate 4 in dry tetrahydrofuran, adding methyl magnesium bromide under the protection of nitrogen, reacting for 0.5-2 hours at room temperature, adding 10% protonic acid to quench the reaction, generating precipitate, and filtering to obtain an intermediate 5; the feeding molar ratio of the intermediate 4 to the methylmagnesium bromide is 1 (3-5), and the protonic acid is selected from one of HClO4, HPF6, HBF4, HCl, HBr, HI, CF3COOH, CF3SO3H and CH3SO 3H;
(1-6) Synthesis of intermediate 6
Mixing the intermediate 5, malonaldehyde diphenylamine hydrochloride and sodium acetate in acetic anhydride, and reacting for 5-8 hours at 80-130 ℃ under the protection of nitrogen; after the reaction is finished, adding ether for precipitation, filtering, dissolving a filter cake by using dichloromethane, and separating by using column chromatography to finally obtain an intermediate 6; wherein the feeding molar ratio of the intermediate 5, the malonaldehyde diphenylamine hydrochloride and the sodium acetate is (1-1.5) to 0.5 (1-1.5).
(1-7) Synthesis of monomer (product 1)
Dissolving the intermediate 6 and the azido polyethylene glycol chain (marked as a compound 5) in dichloromethane, sequentially adding cuprous iodide, nitrogen-nitrogen diisopropylethylamine and acetic acid as catalysts, and stirring at room temperature in a dark place for 8-24 hours. Centrifuging the reaction solution at a high speed, removing the precipitate, concentrating an organic phase, and separating by using a reverse column chromatography to obtain a product 1; wherein the feeding molar ratio of the intermediate 6, the compound 5, cuprous iodide, nitrogen diisopropylethylamine and acetic acid is 1: (1-2.2): (0.01-0.1): (0.01-0.1): (0.01 to 0.1);
(2) the preparation of the NIRlyso-NP nano-assembly comprises two methods:
(2-1) one-step preparation
Dissolving a certain amount of the prepared monomer (NIRlyso) in 1-2 ml of dimethyl sulfoxide organic solvent, ultrasonically dispersing and filtering by using an organic filter membrane with the diameter of 220 micrometers for later use; putting 8-10 ml of deionized water into a clean glass bottle, rapidly and magnetically stirring, rapidly adding the deionized water into the dimethyl sulfoxide solution dissolved with the monomer while stirring, continuously stirring for 4-8 minutes, collecting reaction liquid, and dialyzing for three times to obtain a nano assembly material;
(2-2) two-step preparation (further modification of functional chemical groups)
If the polymer chain end of the nano-assembly is a chemical group capable of further functional modification, such as azido, maleimide group or succinimide group, alkynyl, sulfydryl or amino modified R is further added into the nano-assembly obtained in (2-1)6Stirring overnight at room temperature, and dialyzing to obtain the target modified nano assembly.
The organic nano-assembly (NIRlyso-NP) is in the form of a sphere with a diameter of 40-80 nm.
The maximum absorption peak of the organic nano-assembly (NIRlyso-NP) is between 850nm and 850nm in aqueous solution, phosphate buffer solution and serum, and no emission peak exists.
The organic nano-assembly (NIRlyso-NP) is deaggregated in SDS solution, the maximum absorption peak is positioned at 980nm, and the maximum emission peak is positioned at 1005 nm.
The organic nanometer assembly can be used as a contrast agent for each focus part of a living body, and realizes living body biological imaging with high signal-to-noise ratio and high specificity. For example, the fluorescent probe is used as a near infrared two-zone fluorescent probe for high signal to noise ratio mouse living body imaging.
Drawings
FIG. 1 is an electron micrograph of NIRlyso-NP nano-assemblies.
FIG. 2 is the fluorescence emission spectrum of the NIRlyso-NP nano-assembly in the assembled and disassembled state under the excitation of 808 nm.
FIG. 3 is an image of tumors of 940 challenge, NIRlyso-NP-PEG1000 nano-assemblies on mice.
FIG. 4 is the imaging graph of the 940nm excited NIRlyso-NP-PEG2000-RGD nano assembly body on the detection of mouse arthritis parts.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described with the following embodiments, but the present invention is by no means limited to these examples. The following description is only a preferred embodiment of the present invention, and is only for the purpose of explaining the present invention, and should not be construed as limiting the scope of the present invention. It should be understood that any modification, substitution or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Example 1:
preparing a NIRlyso-NP-PEG1000 nano assembly, wherein the monomer structural formula of the assembly is as follows:
the specific synthetic route is as follows:
the specific synthesis steps are as follows:
(1) synthesis of intermediate 1-1
Dissolving para-bromo substituted thiophenol (compound 1-1) and substituted ethyl phenylacetoacetate (compound 1-2) in polyphosphoric acid, and reacting for 1-3 hours at 90-100 ℃; cooling, adding crushed ice to quench the reaction, extracting with dichloromethane, concentrating an organic phase, and separating by column chromatography to obtain an intermediate 1-1; wherein the feeding molar ratio of the compound 1-1 to the compound 1-2 to the polyphosphoric acid is 1 (1-1.3) to 10-15;
(2) synthesis of intermediate 1-2
Under the protection of nitrogen, mixing the intermediate 1-1, tert-butyloxycarbonyl piperazine, a Buchwald catalyst and an inorganic base in a dry solvent, and reacting for 3-12 hours at 80-110 ℃; cooling to room temperature, filtering, concentrating the organic phase, and separating by column chromatography to obtain intermediate 5-2; wherein the Buchwald catalyst is a composition which is selected from one of palladium acetate and tris (dibenzylideneacetone) dipalladium and one of 2-dicyclohexyl phosphorus-2 ',4',6 '-triisopropyl biphenyl, 4, 5-bis (diphenylphosphino) -9, 9-dimethyl xanthene and 2-dicyclohexyl phosphine-2' - (N, N-dimethylamine) -biphenyl, and the feeding mole percentage of the Buchwald catalyst is 1-10% of that of the intermediate 5-1; the feeding molar ratio of the intermediate 1-1, the tert-butyloxycarbonylpiperazine and the inorganic base is 1 (2-5) to 1.2-3, and the inorganic base is selected from one of sodium tert-butoxide, cesium carbonate, potassium carbonate and potassium phosphate; the solvent is selected from one of toluene, dioxane and tetrahydrofuran;
(3) synthesis of intermediates 1 to 3
Dissolving the intermediate 1-2 in a mixed solvent of dichloromethane and trifluoroacetic acid, stirring for 1-5 hours in an ice bath, and after the reaction is finished, spin-drying the organic solvent to obtain a solid, namely the intermediate 1-3;
(4) synthesis of intermediates 1 to 4
Dissolving the intermediate 1-3, bromopropyne (compound 1-3) and potassium carbonate in acetonitrile, reacting for 10-24 hours at 80 ℃, cooling to room temperature, filtering, concentrating an organic phase, and separating by using column chromatography to obtain an intermediate 1-4; wherein the feeding molar ratio of the intermediate 1-3, the bromopropyne (compound 1-3) and the potassium carbonate is 1: (2-5): (2-5);
(5) synthesis of intermediates 1 to 5
Dissolving the intermediate 1-4 in dry tetrahydrofuran, adding methyl magnesium bromide under the protection of nitrogen, reacting for 0.5-2 hours at room temperature, adding 10% protonic acid to quench the reaction, generating precipitate, and filtering to obtain an intermediate 1-5; wherein the feeding molar ratio of the intermediate 1-4 to the methyl magnesium bromide is 1 (3-5), and the protonic acid is selected from HClO4、HPF6、HBF4、HCl、HBr、HI、CF3COOH、CF3SO3H and CH3SO3H;
(6) synthesis of intermediates 1 to 6
Mixing the intermediate 1-5, malonaldehyde diphenylamine hydrochloride and sodium acetate in acetic anhydride, and reacting for 5-8 hours at 80-130 ℃ under the protection of nitrogen; after the reaction is finished, adding ether for precipitation, filtering, dissolving a filter cake by using dichloromethane, and separating by using column chromatography to finally obtain an intermediate 5-6; wherein the feeding molar ratio of the intermediate 1-5, the malonaldehyde diphenylamine hydrochloride and the sodium acetate is (1-1.5) to 0.5 (1-1.5).
(7) Synthesis of product 1
Dissolving the intermediate 1-6 and the azido polyethylene glycol chain (the compound 1-4) in dichloromethane, sequentially adding cuprous iodide, nitrogen diisopropylethylamine and acetic acid as catalysts, and stirring at room temperature in a dark place for 8-24 hours. The reaction solution was centrifuged at high speed to remove the precipitate, the organic phase was concentrated and separated by reverse column chromatography to give product 1. Wherein the feeding molar ratio of the intermediate 1-6, the compound 1-4, cuprous iodide, nitrogen diisopropylethylamine and acetic acid is 1: (2-10): (0.01-0.1): (0.01-0.1): (0.01-0.1).
(8) Preparation of NIRlyso-NP-PEG1000 Nanocompassembles from product 1
Dissolving a certain amount of the product 1 in 1 ml of dimethyl sulfoxide organic solvent, performing ultrasonic dispersion, and filtering with an organic filter membrane with the diameter of 220 microns for later use; preparing 9 ml of deionized water in a clean glass bottle, rapidly and magnetically stirring, rapidly adding the dimethyl sulfoxide solution dissolved with the monomer while stirring, continuously stirring for five minutes, collecting reaction liquid, and dialyzing for three times.
Example 2:
preparing a NIRlyso-NP-PEG2000-RGD nano assembly, wherein the monomer structural formula of the assembly is as follows:
the specific synthetic route is as follows:
the specific synthesis steps are as follows:
(1) synthesis of intermediate 2-1
Dissolving the intermediate-6 and the azido-polyethylene glycol-succinimide chain (compound 6-1) in dichloromethane, sequentially adding cuprous iodide, nitrogen diisopropylethylamine and acetic acid as catalysts, and stirring at room temperature in a dark place for 8-24 hours. Centrifuging the reaction solution at high speed, removing precipitate, concentrating the organic phase, and separating by reverse column chromatography to obtain intermediate 2-1; wherein the feeding molar ratio of the intermediate 1-6, the compound 2-1, cuprous iodide, nitrogen diisopropylethylamine and acetic acid is 1: (1-1.5): (0.01-0.1): (0.01-0.1): (0.01-0.1).
(2) Preparation of product 2 from intermediate 2-1
Dissolving a certain amount of the intermediate 2-1 in 1 ml of dimethyl sulfoxide organic solvent, performing ultrasonic dispersion, and filtering with an organic filter membrane with the diameter of 220 microns for later use; preparing 9 ml of deionized water in a clean glass bottle, rapidly and magnetically stirring, rapidly adding the dimethyl sulfoxide solution dissolved with the monomer while stirring, continuously stirring for five minutes, collecting reaction liquid, and dialyzing for three times to obtain an assembly of the intermediate 2-1; and (3) continuously adding amino-RGD (compound 2-2) into the solution system, stirring for 6-12 hours at room temperature in a dark place, collecting reaction liquid, and dialyzing for three times to obtain the NIRlyso-NP-PEG2000-RGD nano assembly.
Application example:
the NIRlyso-NP-PEG1000 nano-assembly is used for imaging the epidermoma of the mouse living body. The method comprises the following specific steps:
after tumor-bearing mice were anesthetized, 200. mu.l of Lyso-NIR II-1005 nano-assembly solution was injected into the mice via the tail vein, the mice were irradiated with a 940nm external laser, and the change in the ratio of the fluorescence signal intensity of the tumor region to that of the normal tissue of the mice with time was recorded (see FIG. 3).
The NIRlyso-NP-PEG2000-RGD nano assembly is used for imaging arthritis of a mouse living body. The method comprises the following specific steps:
in a mouse model of arthritis with damaged joints of one leg, 200 microliters of Lyso-NIR II-1005-RGD nano-assembly solution was injected into the body of a mouse through the caudal vein, and the mouse was irradiated with a 940nm external laser to obtain a mouse image in which the focal site of arthritis of the mouse was specifically lighted (see FIG. 4).
Claims (5)
1. An organic nano assembly is a nano material synthesized based on piperazine thiochromene pentamethine cyanine dye structure, is marked as NIRlyso-NP, and a monomer of the organic nano assembly mainly comprises three parts: a series of piperazine benzothiopyran pentamethine cyanine dye structures with luminescent properties, flexible chains connected in the middle and terminal functional groups; the monomer has a general structural formula shown in formula (I):
wherein:
x is selected from O or S, R1～R5Can be independently selected from O (CH)2)n1CH3Or (CH)2)n1CH3Alkyl chains, or can be selected from H, F, Cl, Br, I atoms; r6Is selected from OCH3Succinimide, maleimide, N3RGD or folate functional group, Y is selected from ClO4、PF6、BF4、Cl、Br、I、CF3COO、CF3SO3Or CH3SO3，R7One selected from the group shown in formula (II), wherein n is an integer of 20-50;
2. the method for preparing an organic nano-assembly as claimed in claim 1, characterized by the essential steps of:
(1) substituted thiophenol, substituted ethyl benzoate and propine substituted piperazine are taken as raw materials, and are subjected to polyphosphoric acid condensation cyclization reaction and Buchwald-Hartwig amination reaction sequentially to obtain substituted benzothiopyran derivatives; the benzothiopyran derivative is further alkylated by a methylation format reagent and is treated by acid to prepare a terminal salt, and the terminal salt and a condensing agent of amylene dialdehyde diphenylamine hydrochloride undergo a Knoevenagel condensation reaction to obtain a series of piperazine benzothiopyran pentamethine cyanine dye structures with a propine structure at the tail end and with luminescent properties;
(2) the dye is further connected with a polyethylene glycol chain with one end modified with an azide group through a click chemical reaction to obtain an amphiphilic monomer structure;
(3) the tail end of the monomer structure is further subjected to bioorthogonal reaction to obtain an organic nano assembly;
(4) or further performing an amide reaction to modify the functional biomolecule.
3. The method of claim 2, wherein the specific process comprises (1) the synthesis of a monomer,
the specific synthetic route is as follows:
the specific synthesis steps are as follows:
(1-1) Synthesis of intermediate 1
Dissolving para-bromo substituted thiophenol (marked as a compound 1) and substituted ethyl phenylacetate (marked as a compound 2) in polyphosphoric acid, and reacting for 1-3 hours at 90-100 ℃; cooling, adding crushed ice to quench the reaction, extracting with dichloromethane, concentrating an organic phase, and separating by column chromatography to obtain an intermediate 5-1; wherein the feeding molar ratio of the compound 1 to the compound 2 to the polyphosphoric acid is 1 (1-1.3) to 10-15;
(1-2) Synthesis of intermediate 2
Under the protection of nitrogen, mixing the intermediate 1, tert-butyloxycarbonylpiperazine (marked as a compound 3), a Buchwald catalyst and inorganic base in a dry solvent, and reacting for 3-12 hours at 80-110 ℃; cooling to room temperature, filtering, concentrating the organic phase, and separating by column chromatography to obtain an intermediate 2; wherein the Buchwald catalyst is a composition which is selected from one of palladium acetate and tris (dibenzylideneacetone) dipalladium and one of 2-dicyclohexyl phosphorus-2 ',4',6 '-triisopropyl biphenyl, 4, 5-bis (diphenylphosphino) -9, 9-dimethyl xanthene and 2-dicyclohexyl phosphine-2' - (N, N-dimethylamine) -biphenyl, and the feeding mole percentage of the Buchwald catalyst is 1-10% of that of the intermediate 5-1; the feeding molar ratio of the intermediate 1, the compound 3 and the inorganic base is 1 (2-5) to 1.2-3, and the inorganic base is selected from one of sodium tert-butoxide, cesium carbonate, potassium carbonate and potassium phosphate; the solvent is selected from one of toluene, dioxane and tetrahydrofuran;
(1-3) Synthesis of intermediate 3
Dissolving the intermediate 2 in a mixed solvent of dichloromethane and trifluoroacetic acid, stirring for 1-5 hours in an ice bath, and after the reaction is finished, spin-drying the organic solvent to obtain a solid, namely an intermediate 3;
(1-4) Synthesis of intermediate 4
Dissolving the intermediate 3, bromopropyne (marked as a compound 4) and potassium carbonate in acetonitrile, reacting for 10-24 hours at 80 ℃, cooling to room temperature, filtering, concentrating an organic phase, and separating by using column chromatography to obtain an intermediate 4; wherein the feeding molar ratio of the intermediate 3 to the compound 4 to the potassium carbonate is 1: (2-5): (2-5);
(1-5) Synthesis of intermediate 5
Dissolving the intermediate 4 in dry tetrahydrofuran, adding methyl magnesium bromide under the protection of nitrogen, reacting for 0.5-2 hours at room temperature, adding 10% protonic acid to quench the reaction, generating precipitate, and filtering to obtain an intermediate 5; the feeding molar ratio of the intermediate 4 to the methylmagnesium bromide is 1 (3-5), and the protonic acid is selected from one of HClO4, HPF6, HBF4, HCl, HBr, HI, CF3COOH, CF3SO3H and CH3SO 3H;
(1-6) Synthesis of intermediate 6
Mixing the intermediate 5, malonaldehyde diphenylamine hydrochloride and sodium acetate in acetic anhydride, and reacting for 5-8 hours at 80-130 ℃ under the protection of nitrogen; after the reaction is finished, adding ether for precipitation, filtering, dissolving a filter cake by using dichloromethane, and separating by using column chromatography to finally obtain an intermediate 6; wherein the feeding molar ratio of the intermediate 5, the malonaldehyde diphenylamine hydrochloride and the sodium acetate is (1-1.5) to 0.5 (1-1.5).
(1-7) Synthesis of monomer, product 1
Dissolving the intermediate 6 and an azido polyethylene glycol chain (marked as a compound 5) in dichloromethane, sequentially adding cuprous iodide, nitrogen-nitrogen diisopropylethylamine and acetic acid as catalysts, and stirring at room temperature in a dark place for 8-24 hours; centrifuging the reaction solution at a high speed, removing the precipitate, concentrating an organic phase, and separating by using a reverse column chromatography to obtain a product 1; wherein the feeding molar ratio of the intermediate 6, the compound 5, cuprous iodide, nitrogen diisopropylethylamine and acetic acid is 1: (1-2.2): (0.01-0.1): (0.01-0.1): (0.01 to 0.1);
(2) preparation of organic nano-assembly
Dissolving a certain amount of the prepared monomer in 1-2 ml of dimethyl sulfoxide organic solvent, ultrasonically dispersing, and filtering by using an organic filter membrane with the diameter of 220 microns for later use; and (3) putting 8-10 ml of deionized water into a clean glass bottle, quickly and magnetically stirring, quickly adding the deionized water into the dimethyl sulfoxide solution dissolved with the monomer while stirring, continuously stirring for 4-8 minutes, collecting reaction liquid, and dialyzing for three times to obtain the nano assembly material.
4. The method of claim 3, further modifying functional chemical groups:
for the end of the polymer chain of the nano assembly is a chemical group which can be further functionalized and modified and comprises azido, maleimide group or succinimide group, and alkynyl, sulfydryl or amino modified R is further added into the obtained nano assembly6Stirring overnight at room temperature, and dialyzing to obtain the target modified nano assembly.
5. An application of the organic nano-assembly of claim 1 in-vivo biological imaging with high signal-to-noise ratio of a near-infrared second window based on the combination of fluorescent signal activatability and target recognition.
Priority Applications (1)
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| CN114409890A (en) * | 2022-02-28 | 2022-04-29 | 中国科学院长春应用化学研究所 | Amino-functionalized polyethylene glycol derivative and preparation method thereof |
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